The present proposal is an extension of our previous work aimed at defining the mechanisms that control the proliferation of hepatocytes during late gestation in the rat. During the previous cycle of this project, we showed that expression of the critical proto-oncogene, c-myc, may be regulated by a coding region determinant binding protein (CRD-BP) that stabilizes c-mycmRNA. This finding was consistent with our previous observation that the ERK mitogen-activated protein kinase pathway, known to be involved in transcriptional induction of c-myc, is uncoupled in late gestation fetal liver. We also found that the mitogenic pathway involving phosphotidylinositol-3 kinase (P13K) and ribosomal protein S6 kinase 1 (S6K1) is inactivated in fetal liver, but that an alternative mechanism exists for maintaining the hyperphosphorylation of S6 in fetal hepatocytes. Our most recent studies are consistent with the involvement of an alternative S6K isoform, S6K2, in fetal liver growth. Finally, we demonstrated that mitogenic signaling downstream from insulin is markedly attenuated in late gestation hepatocytes. These findings form the basis for our specific aims. Specific Aim I is to define the mechanisms that regulate the hepatic expression of c-myc during late development, focusing on the role and regulation of CRD-BP. Specific Aim 2 will characterize the alternative pathway that maintains hyperphosphorylation of S6 in late gestation hepatocytes. Our preliminary data indicate that fetal hepatocyte proliferation in vivo is resistant to inhibition by rapamycin, a potent inhibitor of the S6K1 pathway and of liver regeneration. Therefore, we will focus on rapamycin-resistant mechanisms, perhaps those involving S6K2. Specific Aim 3 will focus on the mechanisms for the attenuation of the S6K1 and ERK pathways in developing liver by testing the hypothesis that the actions of inactivating phosphatases in both pathways are modulated by metabolic changes during the perinatal period. We will also examine the mechanism for attenuated insulin signaling, pursuing preliminary data indicating that the primary insulin receptor substrates IRS-1 and IRS-2 are present at very low abundance in developing liver. Finally, in Specific Aim 4 we will determine if the fetal "mitogenic signaling phenotype" defined by our studies is a function of the fetal hepatic environment or is endogenous to fetal hepatocytes. This will be tested in a model of fetal hepatocyte transplantation. We anticipate that our results will provide insight into mechanisms that control the proliferation of hepatocytes under both physiological and pathophysiological conditions.